A comprehensive and integrated program designed to study the membrane transport and enzymology of folate and cobalamin compounds at the molecular level and to obtain information relevant to the design of cancer chemotherapy regimens in described. Four related projects comprise the program: (1) Transport folate compounds including, isolation and characterization of the components of the transport systems; investigation of the transport mechanisms by reconstituting these components in liposomes; development of methods for detecting and quantitating these transport systems in individual cells; and exploitation of the folate transport systems in chemotherapy. (2) Dihydrofolate reductase, including further characterization of multiple forms of the enzyme present in an MTX-resistant subline of L1210 cells; preparation of monoclonal antibodies to the enzyme; and delineation of the mechanism of activation of dihydrofolate reductase. (3) Methionine synthetase and transport of cobalamin compounds, including delineation of the role of cobalamin compounds in the replication of eukaryotic cells; isolation and characterization of the methionine synthetase system from L1210 cells, and further characterization of this system from E.coli K-12; elucidation of the mechanism of transport of cobalamin compounds in eukaryotic cells; and use of the cobalamin transport system for introducing nucleotides into cells. (4) Methenyltetrahydrofolate synthetase, including studies of the structure and mechanism of the enzyme previously purified to homogeneity from L. casei; isolation and characterization of its counterpart from L1210 cells; measurement of the level of the enzyme in various tumor and host cells; and assessment of the role of the enzyme in cell replication and in high dose MTX-folinate rescue regimens. L1210 mouse leukemia cells and Lactobacillus casei will provide the primary experimental systems.